PROJECT SUMMARY Immune checkpoint therapy is proving to be an effective approach for the treatment of a variety of cancers. However, only a subset of patients exhibits long-lasting responses highlighting the critical need for the identification of novel options to augment effects. The nuclear receptor (NR) superfamily of ligand-regulated transcription factors has proven to be an excellent source of targets for therapeutic intervention of a broad range of diseases. The NR2F subfamily of NRs, COUP-TF1 (NR2F1), COUP-TFII (NR2F2), and COUP-TFIII (NR2F6), are considered orphan receptors since their endogenous ligands have yet to be identified. NR2F6 has recently emerged as an intracellular T-cell immune checkpoint; NR2F6-deficient mice spontaneously reject tumors and develop host-protective immunological memory. However, little is known about NR2F6's transcriptional activity, which is due, in part, to lack of specific molecular probes that would enable interrogation of its function. In order to identify ligands that modulate NR2F6 activity, we have designed a high-throughput screening (HTS) compatible primary assay that specifically measures the transcriptional activity of NR2F6 in vitro. Our goal is to implement a full HTS-campaign using the Scripps Institutional Drug Discovery Library (SDDL) to identify, validate, and characterize potent small molecule modulators of NR2F6 activity. To achieve our goal, we have miniaturized our assay into a 1,536-well plate format. Using LOPAC and a 10,000 compound library HTS screen we have validated our assay, meeting HTS automation criteria, and propose to carry out a ?full-deck? HTS campaign to screen the >640,000 compounds in the SDDL (Aim 1). Cheminformatic analysis of ?hits? will help identify the most promising leads by structural clustering, bioinformatics analysis of compounds to determine promiscuity, and scaffold analysis to determine ease of chemical synthesis and tractability for further medicinal chemistry efforts to perform structure-activity relationship studies. In Aim 2, we will use a cascade of follow up assays to validate screening hits, determine specificity, and begin to understand mechanism of action of NR2F6-mediated transcriptional activity. Finally, in Aim 3, validated HTS hits will be advanced to early medicinal chemistry for lead optimization and characterization of novel NR2F6 molecular probes. We expect that completion of this application will deliver multiple structurally distinct NR2F6 modulators that exhibit suitable levels of cellular activity, potency, and selectivity. Further medicinal chemistry efforts will enable development of lead compounds to determine potency and specificity for NR2F6 while minimizing toxicity. Our collaborative research team has a strong track record of performing high-throughput screens, selective optimization of scaffolds, and in vitro and in vivo characterization of compounds. Collectively, our screening approach puts us in a unique position to identify, validate, and characterize novel, NR2F6-selective small molecules for the study of the receptor's function in animal models of disease.